玉米中维生素和植物激素的毛细管液相色谱分离检测
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摘要
高效液相色谱(HPLC)作为一种高效、快速的分离分析技术,在生物工程、环境监测等领域获得广泛的应用。随着分析仪器向小型化发展的趋势,毛细管液相色谱(CLC)越来越多地被人们使用。由于用毛细管柱代替了常规液相柱,CLC节省了样品,减少了有机溶剂的消耗和对环境的污染。与常规液相色谱相比,CLC由于进样量的减小和柱上检测池检测光程的减小导致检测灵敏度的降低,本论文通过扩展检测池的检测光程和溶剂梯度效应以及管内固相微萃取技术(in-tube SPME)提高检测的灵敏度。主要研究内容有:
1.建立了用CLC分离水溶性维生素的方法。采用硅胶基质ODS整体柱(27 cm×100μm I. D.)作为分离柱,以带有光程为3毫米的光纤检测池的紫外检测器作为检测手段,研究了甲醇浓度、缓冲液pH值对分离结果的影响。该方法测定5种水溶性维生素的检测限在14.6-53.5 ng/mL(S/N = 3)范围内,根据迁移时间和峰面积计算得到的迁移时间和峰面积的RSD均小于4.4%,回归方程的线性相关系数在0.9963-0.9994之间。用所建立的方法分析玉米样品,在玉米中未能检测到水溶性维生素,这是由于玉米中水溶性维生素的含量较低,我们所建立方法的灵敏度不够。
2.建立了植物内源激素的CLC测定方法,通过扩展检测池的检测光程和溶剂梯度效应提高检测灵敏度。研究了甲醇浓度、缓冲液pH值、不同样品介质对分离结果的影响。该方法测定4种植物激素的检测限在27.7-196.1 ng/mL(S/N = 3)范围内,根据迁移时间和峰面积计算得到的迁移时间和峰面积的RSD均小于2.8%,回归方程的线性相关系数在0.9984-0.9989之间。用所建立的方法分析玉米样品,在玉米中检测到玉米素(ZT)、赤霉素(GA3)和吲哚-3-乙酸(IAA),并对GA3和IAA进行了定量分析。
3.建立了一种in-tube SPME与CLC在线联用的方法。以毛细管整体柱作为固相萃取柱,对脂溶性维生素及β-胡萝卜素进行萃取富集并在CLC上分离。通过扩展检测池的检测光程和在线in-tube SPME技术提高检测灵敏度。考察了不同流动相、样品基质、进样体积等主要因素对分离和富集结果的影响。该方法测定脂溶性维生素的检测限在1.9 - 176 ng/mL(S/N = 3)范围内。根据迁移时间和峰面积计算得到的迁移时间和峰面积的RSD均小于5.0%,回归方程的线性相关系数在0.9981 - 0.9999之间。用所建立的方法分析玉米样品,在玉米中检测到维生素E(VE),并对其进行了定量分析。
Due to its advantages of high separation efficiency and fast speed, high performance liquid chromatography (HPLC) has been widely applied in the fields of biological detection, environmental monitoring and so on. Miniaturization and automation have been two important trends in LC field, and micro or capillary liquid chromatography (CLC) has been used more and more. Compared with conventional LC, micro or capillary LC offers several advantages such as significant reduction of solvent consumption, small amounts of sample required. However, a minute sample injection volume and limited optical path length for on-column UV photometric detection in CLC result in its low detection sensitivity. In order to enhance the detection sensitivity in CLC, hyphenation with sample preconcentration techniques is needed. In this work, some on-column focusing technique such as in-tube solid-phase microextraction (SPME) and solvent gradient zone sharpening effect were investigated to enhance the detection sensitivity. The major content is described as follows:
1. A sensitive CLC method has been proposed for the determination of water-soluble vitamins. An optical-fiber flow cell with a long light path in the UV detector was used to improve the detection sensitivity. The effects of some parameters on the separation (including methanol concentration and pH of mobile phase) were investigated. The detection limits (LODs) of the water-soluble vitamins are in the range from 14.6 to 53.5 ng/mL based on the signal-to-noise ratio of 3 (S/N = 3). The relative standard deviations (RSDs) of migration time and peak area for each water-soluble vitamins were less than 4.4%. The developed method was applied to the analysis of water-soluble vitamins in corns. Water-soluble vitamins were not detected in corn samples since the detection sensitivity of the developed method for water-soluble vitamins was not high enough to detect them due to their low contents in corns.
2. A CLC method was developed for the analysis of phytohormones. An optical-fiber flow cell with a long light path in the UV detector and solvent gradient zone sharpening effect were combined to enhance the detection sensitivity. Some parameters, such as methanol concentration and sample matrix, were optimized. The LODs of the phytohormones are in the range from 27.2 to 196.1 ng/mL based on the signal-to-noise ratio of 3 (S/N = 3). The RSDs of migration time and peak area for each phytohormone were less than 2.8%. The method was applied to the analysis of phytohormones in different types of corns. Zeatin (ZT), gibberellin A3 (GA3) and indole-3-acetic acid (IAA) in corn samples were detected, in which GA3 and IAA were analyzed quantitatively.
3. A CLC system with UV/Vis detection was coupled with an in-tube SPME device for the analysis of fat-soluble vitamins andβ-carotene. A monolithic silica-ODS column was used as the extraction medium. An optical-fiber flow cell with a long light path in the UV/Vis detector and solvent gradient zone sharpening effect were utilized to further enhance the detection sensitivity. In the in-tube SPME/CLC system, the pre-condition of the extraction column and the effect of the injection volume were studied. The LODs for the fat-soluble vitamins andβ-carotene were in the range from 1.9 to 173 ng/mL based on the signal-to-noise ratio of 3 (S/N = 3). The RSDs of migration time and peak area for each analyte were less than 5.0%. The method was applied to the analysis of fat-soluble vitamins andβ-carotene contents in different types of corns.α-tocopherol (VE) in corn sample was detected and analyzed quantitatively.
1.建立了用CLC分离水溶性维生素的方法。采用硅胶基质ODS整体柱(27 cm×100μm I. D.)作为分离柱,以带有光程为3毫米的光纤检测池的紫外检测器作为检测手段,研究了甲醇浓度、缓冲液pH值对分离结果的影响。该方法测定5种水溶性维生素的检测限在14.6-53.5 ng/mL(S/N = 3)范围内,根据迁移时间和峰面积计算得到的迁移时间和峰面积的RSD均小于4.4%,回归方程的线性相关系数在0.9963-0.9994之间。用所建立的方法分析玉米样品,在玉米中未能检测到水溶性维生素,这是由于玉米中水溶性维生素的含量较低,我们所建立方法的灵敏度不够。
2.建立了植物内源激素的CLC测定方法,通过扩展检测池的检测光程和溶剂梯度效应提高检测灵敏度。研究了甲醇浓度、缓冲液pH值、不同样品介质对分离结果的影响。该方法测定4种植物激素的检测限在27.7-196.1 ng/mL(S/N = 3)范围内,根据迁移时间和峰面积计算得到的迁移时间和峰面积的RSD均小于2.8%,回归方程的线性相关系数在0.9984-0.9989之间。用所建立的方法分析玉米样品,在玉米中检测到玉米素(ZT)、赤霉素(GA3)和吲哚-3-乙酸(IAA),并对GA3和IAA进行了定量分析。
3.建立了一种in-tube SPME与CLC在线联用的方法。以毛细管整体柱作为固相萃取柱,对脂溶性维生素及β-胡萝卜素进行萃取富集并在CLC上分离。通过扩展检测池的检测光程和在线in-tube SPME技术提高检测灵敏度。考察了不同流动相、样品基质、进样体积等主要因素对分离和富集结果的影响。该方法测定脂溶性维生素的检测限在1.9 - 176 ng/mL(S/N = 3)范围内。根据迁移时间和峰面积计算得到的迁移时间和峰面积的RSD均小于5.0%,回归方程的线性相关系数在0.9981 - 0.9999之间。用所建立的方法分析玉米样品,在玉米中检测到维生素E(VE),并对其进行了定量分析。
Due to its advantages of high separation efficiency and fast speed, high performance liquid chromatography (HPLC) has been widely applied in the fields of biological detection, environmental monitoring and so on. Miniaturization and automation have been two important trends in LC field, and micro or capillary liquid chromatography (CLC) has been used more and more. Compared with conventional LC, micro or capillary LC offers several advantages such as significant reduction of solvent consumption, small amounts of sample required. However, a minute sample injection volume and limited optical path length for on-column UV photometric detection in CLC result in its low detection sensitivity. In order to enhance the detection sensitivity in CLC, hyphenation with sample preconcentration techniques is needed. In this work, some on-column focusing technique such as in-tube solid-phase microextraction (SPME) and solvent gradient zone sharpening effect were investigated to enhance the detection sensitivity. The major content is described as follows:
1. A sensitive CLC method has been proposed for the determination of water-soluble vitamins. An optical-fiber flow cell with a long light path in the UV detector was used to improve the detection sensitivity. The effects of some parameters on the separation (including methanol concentration and pH of mobile phase) were investigated. The detection limits (LODs) of the water-soluble vitamins are in the range from 14.6 to 53.5 ng/mL based on the signal-to-noise ratio of 3 (S/N = 3). The relative standard deviations (RSDs) of migration time and peak area for each water-soluble vitamins were less than 4.4%. The developed method was applied to the analysis of water-soluble vitamins in corns. Water-soluble vitamins were not detected in corn samples since the detection sensitivity of the developed method for water-soluble vitamins was not high enough to detect them due to their low contents in corns.
2. A CLC method was developed for the analysis of phytohormones. An optical-fiber flow cell with a long light path in the UV detector and solvent gradient zone sharpening effect were combined to enhance the detection sensitivity. Some parameters, such as methanol concentration and sample matrix, were optimized. The LODs of the phytohormones are in the range from 27.2 to 196.1 ng/mL based on the signal-to-noise ratio of 3 (S/N = 3). The RSDs of migration time and peak area for each phytohormone were less than 2.8%. The method was applied to the analysis of phytohormones in different types of corns. Zeatin (ZT), gibberellin A3 (GA3) and indole-3-acetic acid (IAA) in corn samples were detected, in which GA3 and IAA were analyzed quantitatively.
3. A CLC system with UV/Vis detection was coupled with an in-tube SPME device for the analysis of fat-soluble vitamins andβ-carotene. A monolithic silica-ODS column was used as the extraction medium. An optical-fiber flow cell with a long light path in the UV/Vis detector and solvent gradient zone sharpening effect were utilized to further enhance the detection sensitivity. In the in-tube SPME/CLC system, the pre-condition of the extraction column and the effect of the injection volume were studied. The LODs for the fat-soluble vitamins andβ-carotene were in the range from 1.9 to 173 ng/mL based on the signal-to-noise ratio of 3 (S/N = 3). The RSDs of migration time and peak area for each analyte were less than 5.0%. The method was applied to the analysis of fat-soluble vitamins andβ-carotene contents in different types of corns.α-tocopherol (VE) in corn sample was detected and analyzed quantitatively.
引文
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